Method for the quantification of pd-l1 expression

ABSTRACT

A highly sensitive for determining the expression of PD-L1 that is based on a RT-qPCR in a RNA sample of, for example, Circulating Tumor Cells (CTC) or fresh frozen primary tumor tissues. In particular, to a method for the detection of PD-L1 mRNA positive CTCs or primary tumor tissues (fresh frozen) based on the quantitative determination of the molecular marker (PD-L1) in biological samples of patients suffering from cancer. By using the method, detection can take place before, during or after the immune therapy or any other treatment in order to provide significant information concerning guiding or monitoring of the anti-PD-L1 agents effectiveness. This RT-qPCR assay could comprise a promising companion diagnostic test in order to evaluate the PD-L1 expressional status on CTC or tumor tissue, providing clinical applications, which could have an important impact on therapeutic interventions since the expression of PD-L1 is associated with response to immunotherapy.

TECHNICAL FIELD

The present invention relates to a highly sensitive, specific andreproducible real time RT-qPCR assay for the quantification of PD-L1expression in a RNA sample, such as an isolated RNA sample fromCirculating Tumor Cells (CTCs) in peripheral blood of patients withsolid cancers or fresh frozen primary tumor tissues.

BACKGROUND OF THE INVENTION

The discovery of crucial molecular pathways that promote tumor growthand maintenance together with the development of drugs that specificallyinhibit these pathways has changed much in the perennially limitedpanoply of options to treat many types of cancer. The promise of theemerging field of personalized medicine will increasingly be used totailor therapeutics to defined sub-populations, and eventually,individual patients in order to enhance efficacy and minimize adverseside effects. However, the success of personalized medicine depends onhaving accurate, reproducible and clinically useful companion diagnostictests to identify patients who can benefit from targeted therapies.According to this concept, the most recent paradigm is the successfulco-development of a first-in-class selective inhibitor of oncogenic BRAFkinase, vemurafenib (ZELBORAF), which targets only the mutant BRAFprotein, which is detected V600E positive by the approved companiondiagnostic test, the Cobas 4800 BRAF V600 mutation test (Roche MolecularDiagnostics).

Despite the recent development of targeted therapies in many types ofcancer, many patients do not benefit from these therapies. This fact ledto an improved understanding of the mechanisms of protective anti-tumorimmunity and the development of more efficacious immunotherapies thatincrease patient survival confirming the long-standing idea thatimmunity plays an important role in cancer pathogenesis. Immunotherapyis a type of cancer treatment designed to boost the body's naturaldefenses to fight cancer. It uses materials either made by the body orin a laboratory to improve, target, or restore the function of theimmune system. It is now evident through intense research efforts thatcancer cells can develop specific mechanisms to avoidimmunosurveillance. One of these mechanisms is to present itself to theimmune system in such a way that it fails to recognize it as somethingthat should be killed. Another mechanism is to interfere with theabilities of T-cells, whose duty it is to carry out such killings andwhich, by hanging around for decades in the body, provide durableimmunity to a given disease. Lastly, there are all sorts of ways inwhich the immune system as a whole can be suppressed.

Human cancers are characterized by high frequencies of genetic andepigenetic alterations, generating neo-antigens potentially recognizableby the immune system. The ability of the immune system to recognise itsnormal cells from tumor cells and to attack against foreign invaderssuch as viruses provides the basis for the rapid and specific clearanceof most infections. The immune system is able to detect and eliminatemost tumors, which are at an early stage and clinically non-detectableby the concept of cancer immuno-editing. However, the cancer cells use a“cellular camouflage” in order to fool the immune system into thinkingthat they are normal cells; as a result some tumors are not completelydestroyed and escape from the immune-surveillance. A basic hindrance forimmunotherapeutic approaches is that the majority of mechanisms areactive at the tumor site, which act together in order to balanceeffectively the anti-tumor immunity.

Despite the fact that an endogenous immune response to cancer has beenobserved in patients, this response seems to be ineffective, and theestablished cancers are tolerated by the immune system. Contributing tothis state of tolerance, tumors may exploit several distinct mechanismsto actively suppress the host immune response. Among these mechanisms,endogenous “immune checkpoints” that normally terminate immune responsesto mitigate collateral tissue damage can be co-opted by tumors to evadeimmune destruction. Recently, many specific immune checkpoint pathwayinhibitors have been developed and approved by FDA to provide newimmunotherapeutic approaches for treating cancer, including thedevelopment of the anti-CTLA-4 antibody (Ab), ipilimumab (Yervoy), forthe treatment of patients with advanced melanoma and anti-PD-L1antibody, nivolumab (Opdivo), for the treatment of patients withadvanced squamous non-small cell lung cancer (NSCLC) who have progressedon or after platinum-based chemotherapy.

Programmed death 1 (PD-1) is an immune inhibitory receptor expressed onseveral immune cells, particularly cytotoxic T cells. This receptorinteracts with two ligands, programmed death ligand 1 (PD-L1) (B7-H1,CD274) and PD-L2 (B7-DC). The PD-1 has greater affinity for PD-L1, whichis widely expressed on tumor cells, as well as other immune cells, whilePD-L2 is expressed primarily on macrophages and dendritic cells [Keir M,Butte M, Freeman G, Sharpe A. PD-1 and its ligands in tolerance andimmunity. Annu Rev Immunol 2008; 26: 677-704]. When PD-1 binds to tumorcells expressing PD-L1, T-cell activity and cytokine production aresuppressed, leading to T-cell exhaustion. PD-L1 ligation with PD-1during infection or inflammation in normal tissues is criticallyimportant in maintaining homeostasis of immune response to preventautoimmunity. The PD-1/PD-L1 interaction in the tumor microenvironment,however, provides an immune escape for tumor cells by turning offcytotoxic T cells. Thus by blocking this interaction, it is expectedthat tumor cells will be attacked by cytotoxic T cells. The activationof the PD-1/PD-L1 pathway protects tumor cells from immunologicalresponses mediated by T-cells. Inhibition of the PD-1/PD-L1 pathwayreverses immune evasion by replenishing a plethora of activatednon-exhausted T-cells. The development and clinical testing of immuneblocking antibodies has resulted in until now clinical activity in avariety of malignancies including melanoma and lung cancer [Callahan MK, Wolchok J D. At the bedside: CTLA-4 and PD-1-blocking antibodies incancer immunotherapy. J Leukoc Biol. 2013; 94: 41-53].

Most recently, Larkin et al., presented a randomized, double-blind,phase 3 study, in which the nivolumab (anti-PD-1 antibody) asmonotherapy or the combination nivolumab plus ipilimumab (anti-CTLA-4antibody) were compared to ipilmumab alone in patients with metastaticmelanoma. The median progression-free survival was longer with nivolumabplus ipilimumab, as compared with ipilimumab or nivolumab asmonotherapy. However, in patients with PD-L1-positive tumors, the medianprogression-free survival (PFS) was 14.0 months in thenivolumab-plus-ipilimumab group and in the nivolumab group, but inpatients with PD-L1-negative tumors, PFS was longer with the combinationtherapy than with nivolumab alone. From these results it has becomeclear that the expression of PD-L1 seems to be a significant predictivebiomarker in anti-PD-L1 treatments. Thus the need for developing a newcompanion diagnostic test for detecting PD-L1 positive or negativetumors, is increasing [Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey C L, Lao C D, et al. Combined Nivolumab and Ipilimumab orMonotherapy in Untreated Melanoma. N Engl J Med. 2015; 373: 23-34].

Recently, the “liquid biopsy” approach has a high potential to changesignificantly the therapeutic strategy in patients suffering from manytypes of cancer. The detection, enumeration and the molecularcharacterization of Circulating Tumor Cells (CTCs), cell-freecirculating tumor DNA (ctDNA) and circulating microRNAs (miRNAs) providean extremely powerful and reliable non-invasive source of blood-basedbiomarkers for the individual molecular profiling of each patient inreal time, and especially before and after treatment. The applicationsof liquid biopsy are based on the identification of molecular targets,the assessment of prognosis, the diagnosis of recurrence/progression,the monitoring of response to therapy and monitoring of tumor genomicprofiles in real time. Most importantly, these blood-based tests seem tobe very challenging and highly important in case that tumor biopsies arenot accessible (such as in the case of NSCLC). Moreover they can offer aclose follow-up of disease biomarkers enabling the monitoring of theefficacy of treatment and potentially improve the choice of treatmentoptions.

Recently, Mazel et al., using the CellSearch™ system (Veridex, J&J), anFDA-cleared platform for CTCs enumeration, by using an anti-PD-L1specific antibody and the fourth channel for imaging in the CellSearch,found PD-L1 positive CTCs in 11 out of 16 (68.8%) patients withmetastatic breast cancer. More specifically, in their study, they haveused the anti-human 87-H1/PD-L1PE-conjugated monoclonal antibody forPD-L1 expression, suggesting that this assay could be used for liquidbiopsy for monitoring and stratification of cancer patients undergoingimmune checkpoint blockade [Mazel M, Jacot W, Klaus P, Bartkowiak K,Topart D, Cayrefourcq L et al. Frequent expression of PD-L1 oncirculating breast cancer cells. Mol Oncol. 2015, Article in press].Moreover, Oliveira-Costa et al., found a strong cytoplasmatic expressionof PD-L1 in CTCs in oral squamous cell carcinoma usingimmunofluorescence and Nanostring [Oliveira-Costa J P, Fiorini deCarvalho A, Gobbi da Silveira G, Amaya P, Wu Y et al., Gene expressionpatterns through oral squamous cell carcinoma development: PD-L1expression in primary tumor and circulating tumor cells. Oncotarget.2015; 6: 20902-20].

The detection of PD-L1 expression in CTCs in a quantitative way could beused for monitoring the efficacy of immune checkpoint inhibitors as a“liquid biopsy” approach and could offer many advances in clinicalpractice. Thus, for this reason, an ultrasensitive and highly specificRT-qPCR based method for the quantification of PD-L1 expression in CTCswas developed and validated, suitable for monitoring the efficacy ofcheckpoint inhibitors in peripheral blood of cancer patients. This assayhas a very high analytical sensitivity and specificity in order toquantify the expression of PD-L1 in CTCs, and is highly sensitive incomparison to other methods used in tumor tissues such asimmunohistochemistry. Moreover, the developed method is a closed tubemolecular diagnostic test that comprises a simple and high throughputclinical tool, which can easily be automated and used in most moleculardiagnostic clinical laboratories since it does not require expensiveinstrumentation, and is easy to use. Moreover, the developed method isquantitative, thus enabling not only to recognize differences in theexpression of PD-L1 in clinical samples, but moreover to be subjected todaily quality control performance tests (robustness, reproducibility,within-run and between-run precision) that are very important foraccreditation purposes.

For the first time a novel RT-qPCR based method with novel specificallydesigned primers and a hydrolysis probe for the quantification of PD-L1mRNA transcripts in a human clinical sample and especially in peripheralblood (CTCs) was designed. Especially for CTCs molecularcharacterization, highly sensitive, robust and specific methodologiesare needed. Here it is provided evidence that this assay is suitable forselecting patients for immunotherapy based on the administration ofcheckpoint inhibitors, as well as for monitoring the efficacy ofcheckpoint inhibitors in peripheral blood of cancer patients.

SUMMARY OF THE INVENTION

It is an object to provide an improved method for quantifying PD-L1 mRNAexpression in a RNA sample such as an isolated RNA sample of CTCs inperipheral blood or fresh frozen tumor tissues of patients with solidtumors. The object is wholly or partially achieved by a method accordingto claim 1. Embodiments and further details of the invention are set offorth in the appended dependent claims, in the drawings and in thesequence listing. In particular the invention relates to an in vitromethod for quantitative determination of the expression of ProgrammedDeath Ligand 1 (PD-L1) mRNA in a sample, said method comprising thesteps:

-   -   i. subjecting the sample to reverse transcription using RNA        present in the sample as a template in order synthesize a        corresponding cDNA sequence,    -   ii. forming a reaction mixture comprising the sample, nucleic        acid amplification reagents, a target primer pair, a target        hydrolysis probe, said target primer pair and target hydrolysis        probe being capable of hybridizing to PD-L1 mRNA,    -   iii. subjecting the reaction mixture to amplification conditions        optimized to generate at least one copy of a nucleic acid        sequence complementary to a target sequence, said target        sequence being a mRNA transcript of the PD-L1 mRNA sequence (SEQ        ID NO: 1), and/or    -   iv. determining the amount of PD-L1 mRNA in a said sample,    -   v. normalizing the expression of PD-L1 with respect to an        expression of a reference gene, and    -   vi. comparing the amount of PD-L1 mRNA expressed in a said        sample to a positive and negative control in order to estimate        an overexpression of the PD-L1 mRNA sequence.

The method is based on the quantitative determination of PD-L1 mRNAexpression in a sample. This assay may comprise a downstream analysis ofPD-L1 mRNA expression in a RNA sample, such as an isolated RNA sample ofCTCs in peripheral blood or fresh frozen tumor tissues of patients withsolid cancers.

The CTCs isolation step could be performed by methods known to theskilled person, such as e.g. different microfluidic and filtration bysize devices, density gradient centrifugation, positive and negativeimmunomagnetic selection, the CellSearch™ device, single cell analysissystems, or in-vivo CTC isolation systems such as the cell collectorsystem (Gilupi, GmbH) or leukapheresis systems. It should be noted thatCTC isolation is not limited to the listed methods.

In particular the target sequence in the method described herein is themRNA-transcript of the PD-L1 gene, which is derived after reversetranscription of the RNA present in the sample by using a commerciallyavailable cDNA kit.

The quantification of PD-L1 mRNA expression may be used in many types ofcancer such as breast, urothelial, colorectal, oesophageal, gastric,hepatocellular carcinoma, lung, melanoma, oropharyngeal squamous cellcarcinoma, nasopharyngeal, multiple myeloma, renal cell carcinoma,cervical, glioblastoma, malignant mesotheliomas, lymphomas, ovarian andpancreatic. Advantageously, the quantification of PD-L1 mRNA expressionmay be used in any type of malignant neoplastic disease in order toquantify an expression of PD-L1 mRNA.

The method according to the invention amplifies specifically only thePD-L1 mRNA sequence, avoiding the amplification of genomic DNA. Themethod comprises a RT-qPCR, using a target primer pair, which comprisesat least one intron-spanning site and a target hydrolysis probe.Advantageously the intron-spanning site may comprise only one base ateither site of the intron resulting in that the target primers only bindto a sequence without introns under the conditions employed. The methodutilizes a target primer pair that will only bind to a sequence in whichthe introns have been spliced-out, e.g. mRNA, cDNA. Moreover, the targethydrolysis probe is part of the amplification reaction mixture andhybridizes to the newly synthesized target sequence under selectconditions, provided that this target sequence is present in the testsample.

According to the invention the main feature of the target primer pair isthat it comprises at least one intron-spanning site. This provides atarget primer pair that will only bind to a sequence in which theintrons have been spliced out, e.g. mRNA, cDNA. It should be understoodthat said “splicing” may occur naturally i.e. to provide for thedetection of mRNA in a biological sample. However, the term alsoencompasses an engineered sequence having the introns “spliced out” ofthe sequence, e.g. cDNA. The intron-spanning site may comprise only onebase at either site of the intron provided that the target primer onlybinds the sequence without introns under the conditions employed. One orboth of the forward and reverse target primers may comprise one or moreintron-spanning site(s). In a preferred embodiment both target primerscomprise an intron-spanning site.

In particular, the forward target primer is the primer that is extendedin the same direction as the coding strand of the target nucleic acid.The forward target primer is designed to hybridize between exons 4 and 5of the PD-L1 mRNA sequence in order to enhance only the mRNA targetsequence avoiding DNA genomic amplification. The forward target primeris complementary to the 3′-end. Conversely, the reverse target primer isthe primer that is extended in the same direction as the non-codingstrand of the target nucleic acid. The reverse target primer is designedto hybridize between exons 5 and 6 of the PD-L1 mRNA sequence foramplification of only the target sequence avoiding non-specificproducts. Consequently, the target primers align with their 3′-endsfacing each other.

More particularly, the “target primer pair”, which is used in themethod, is capable of hybridizing to a sequence of PD-L1 mRNA.

In particular the target sequence is the mRNA-transcript of the PD-L1mRNA sequence (SEQ ID NO: 1). It should be noted that the DNA sequencefor PD-L1 (SEQ ID NO: 1) listed in the enclosed sequence listingcorresponds to the transcribed mRNA sequence of said protein. In apreferred embodiment the forward target primer should include at leastthe sequence 5′-TCATCCCAGAA-3′ (SEQ ID NO: 3), more preferably theforward target primer includes the sequence 5′-GCTGAATTGGTCATCCCAGAA-3′(SEQ ID NO: 4). In a preferred embodiment the reverse target primershould include at least the sequence 5′-CATTCTCCCTT-3′ ID NO: 5), morepreferably the reverse target primer includes the sequence5′-TTTCACATCCATCATTCTCCCTT-3′ (SEQ ID NO: 6). The invention furthermorediscloses sequences of specific probes, such as target hybridizationprobes, hydrolysis (TaqMan) probes or molecular Beacon, or SCORPION typeprobes, for detection/quantification of the amplification PCR product.Furthermore, the probe may be used to ensure specificity. Said probe isa target hydrolysis probe. Preferably the target hydrolysis probe shouldinclude at least the sequence 5′-GCACATCCTCCA-3′ (SEQ ID NO: 7), morepreferably the target hydrolysis probe includes, the sequence5′-6FAM-ACCTCTGGCACATCCTCCAAATGAAAG-BBQ-3′ (SEQ ID NO: 8) and twofluorescent particles. The probes are preferably labelled. The label canbe either directly detectable with for example fluorophores,chemiluminophores, fluorescent particles and the like or indirectlydetectable with specific binding partners and nucleic acids. Preferredlabels are directly detectable, and particular preferred labels arefluorescent dyes, such as SYBR Green I, FAM, HEX, VIC, fluorescein LCRed 610, LC Red640, LC Red670, LC Red 705, and other fluorescent dyesknown in the art.

In particular, the target hydrolysis probe is designed to hybridize toan internal region within the amplicon. Especially, the targethydrolysis probe which is used in this method is designed to hybridizeto an internal region of exon 5 of the PD-L1 mRNA sequence. It istypically a 20-30 bp oligonucleotide with a fluorescent reporter dye(i.e. 6-fluorescein, FAM) covalently attached to the 5′ end and afluorescent quencher dye (i.e. BlackBerry Quencher, BBQ). The proximallylocated quencher dye reduces the emission intensity of the reporter dye.The hydrolysis probe is added directly to the PCR mix, and conditionsare virtually identical to those that are established for a standardPCR. During the extension phase of the PCR cycle, the Taq DNA polymerasecleaves the hydrolysis probe only when it is hybridized to the target,separating the reporter dye from the quencher dye. An increase influorescence intensity at 518 nm (when FAM is the reporter dye) (due tothe release of the quenching effect on the reporter) is the result ofhydrolysis probe hydrolysis and is quantitative for the initial amountof the template. The fluorescence intensity specific to the reporter dyeincreases because of its lack of proximity to the quencher dye. Repeatedcycles of denaturation, annealing, and extension result in exponentialamplification of the PCR product and of fluorescence intensity.

In another aspect, step (v) of the method may further comprise areference primer pair that hybridizes to a reference mRNA sequence of areference gene in order to ensure that amplifiable material is presentin the test samples and in order to avoid false negative results.

A reference gene ideally should be stable, expressed in the cells andtissues of interest that do not show changes under the experimentalconditions or disease state. These genes are used to normalize the mRNAlevels of genes of interest before the comparison between differentsamples by the RT-qPCR. These reference genes are responsible formeasuring and reducing the errors from variations among the samples,extraction and RNA quality and efficiency in cDNA synthesis, internalcontrols and the different experimental samples like in normal cells ortumor tissue. In the RT-qPCR method, an appropriate reference geneshould be considered for accurate quantification of mRNA expressionbecause the quantification cycle (C_(Q)) of the target genes is comparedto the C_(Q) of the reference gene. The expression levels of thereference gene should remain constant between different cells types;otherwise the normalization to varying internal reference can result toincreased errors. Several genes have been used as reference genes,including hypoxanthine phosphoribosyl transferase (HPRT),β2-microglobulin (B2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)and β-actin (ACTB), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28SrRNA), α-tubulin (TUBA), albumin (ALB), ribosomal protein L32 (RPL32),TATA sequence binding protein (TBP), cyclophilin C (CYCC), Eelongationfactor 1α (EF1A), RNA polymerase II (RPII) [Suzuki T, Higgins P J,Crawford D R. Control selection for RNA quantitation. Biotechniques,2000; 29:332-7].

In a preferred embodiment, B2M used as a reference gene in thisinvention, and advantageously said reference primer pair was designed tohybridize to the B2M (Beta-2-Microglobulin) mRNA sequence (SEQ ID NO:2).

Therefore, in another aspect of the invention provides a referenceprimer pair of the B2M mRNA sequence (SEQ ID NO: 2). It should be notedthat the DNA sequence B2M (SEQ ID NO: 2) listed in the enclosed sequencelisting corresponds to the transcribed mRNA sequence of said protein. Ina preferred embodiment the forward reference primer should include atleast the sequence 5′-GCCGTGTGAAC-3′ (SEQ ID NO: 9), more preferably theforward reference primer includes sequence 5′-GCCTGCCGTGTGAACCATGT-3′(SEQ ID NO: 10). In a preferred embodiment the reverse reference primershould include at least the sequence 5′-CTTCAAACCTC-3′ (SEQ ID NO: 11),more preferably the reverse reference primer includes the sequence5′-AAATGCGGCATCTTCAAACCTC-3′ (SEQ ID NO: 12).

In the context of the present invention “reference primer pair” and“target primer pair” are not the same. In the context of the presentinvention the term “reference primer pair” is intended to mean a primerpair, which is capable of hybridizing to a sequence of a gene, which isubiquitous to a given cell. More particularly, the “reference primerpair”, which is used in the method, is capable of hybridizing to asequence of B2M mRNA sequence. In other words a “reference primer pair”can be used as an internal control in a method or kit of the invention.

In Real-time RT-qPCR a hybridization probe for the quantification of B2Mexpression may be used as described previously in relation to the PD-L1primer pair. In a preferred embodiment the reference hydrolysis probeshould include at least the sequence 5′-CTCGATCCCAC-3′ (SEQ ID NO: 13),more preferably the target hydrolysis probe includes the sequence5′-6FAM-CATGATGCTGCTTACATGTCTCGATCCCAC-BBQ-3′ (SEQ ID NO: 14) and twofluorescent particles.

In further aspect the present document also relates to a method for:

-   -   i. diagnosing and/or prognosing malignant neoplastic disease in        a subject before immunotherapy with checkpoint inhibitors,        and/or    -   ii. predicting efficacy of treatment of malignant neoplastic        disease in a subject before immunotherapy with checkpoint        inhibitors, and/or    -   iii. assessing outcome of treatment of malignant neoplastic        disease in a subject during and after immunotherapy with        checkpoint inhibitors, and/or    -   iv. assessing the recurrence of malignant neoplastic disease in        a subject during and after immunotherapy with checkpoint        inhibitors,        wherein said method comprises the quantification of the PD-L1        mRNA in a sample according to the steps for the in vitro method        as described herein,        wherein the subject is a human being, who suffers from a        malignant neoplastic disease,        wherein the checkpoint inhibitors may comprise the immune        checkpoint blockade inhibitors, such as anti-PD-L1 inhibitors.

The in vitro method as described herein may advantageously be used fordetermining the amount of the PD-L1 mRNA in a sample for:

-   -   i. diagnosing and/or prognosing malignant neoplastic disease in        a subject before immunotherapy with checkpoint inhibitors,        and/or    -   ii. predicting efficacy of treatment of malignant neoplastic        disease in a subject before immunotherapy with checkpoint        inhibitors, and/or    -   iii. assessing outcome of treatment of malignant neoplastic        disease in a subject during and after immunotherapy with        checkpoint inhibitors, and/or    -   iv. assessing the recurrence of malignant neoplastic disease in        a subject during and after immunotherapy with checkpoint        inhibitors,        wherein the sample analyzed may be a biological sample, and said        biological sample may be obtained from a subject.        Advantageously, the subject is a mammal such as a human being,        who suffers from a malignant neoplastic disease.

The malignant neoplastic disease may be selected from the groupconsisting of breast, urothelial, colorectal, oesophageal, gastric,hepatocellular carcinoma, lung, nasopharyngeal, multiple myeloma, renalcell carcinoma, lymphomas, melanoma, oropharyngeal squamous cellcarcinoma, cervical, glioblastoma, malignant mesotheliomas, ovarian andpancreatic cancer.

The in vitro method as described herein is advantageously used when thesubject suffers from breast and non-small cell lung cancer.

When diagnosing and/or prognosing malignant neoplastic disease in asubject before immunotherapy with checkpoint inhibitors, the methodcomprises the steps of:

-   -   a) performing the in vitro method for quantification of the        PD-L1 mRNA in a sample as described herein,    -   b) determining the amount of PD-L1 mRNA in a said sample,    -   c) normalizing the expression of PD-L1 with respect to B2M        expression, used as a reference gene,    -   d) comparing the amount of PD-L1 mRNA detected in a said sample        to a positive control and a negative control in order to        estimate the over-expression of PD-L1, thereby deriving        prognostic information.

The sample is advantageously obtained from a subject who is sufferingfrom a malignant neoplastic disease and who has not been prescribedimmunotherapy with checkpoint inhibitors. Further embodiments arewherein the positive control comprises peripheral blood mononuclearcells (PBMC) from healthy control samples since PBMC express as wellPD-L1 at very low levels.

Thus, an indication of prognostic information is a relative change inthe amount of PD-L1 mRNA in a said sample that identifies beforeimmunotherapy with checkpoint inhibitors and may estimate the risk offuture outcomes in an individual based on their clinical andnon-clinical characteristics. In particular, a method of determining theprognosis as used herein refers to the prediction of the outcome of, orfuture course of, a subject. Prognosis includes the prediction ofpatient's survival. Moreover, prognosis may be used to predict thedisease free survival time of an individual, progression-free survivaltime, disease specific survival time, survival rate, or survival time.Prognostic testing may be undertaken with (e.g. at the same time as) thediagnosis of a previously undiagnosed cancerous condition, or may relateto an existing (previously diagnosed) condition.

When predicting efficacy of treatment of malignant neoplastic disease ina subject before immunotherapy with checkpoint inhibitors, the methodcomprises the steps of:

-   -   a) performing the in vitro method for quantification of the        PD-L1 mRNA in a sample as described herein,    -   b) determining the amount of PD-L1 mRNA in a said sample,    -   c) normalizing the expression of PD-L1 with respect to B2M        expression, used as a reference gene,    -   d) comparing the amount of PD-L1 mRNA detected in a said sample        to a positive and a negative control in order to estimate the        overexpression of PD-L1, thereby predicting efficacy of        treatment of malignant neoplastic disease in a subject.

The sample is advantageously obtained from a subject who is sufferingfrom a malignant neoplastic disease and who has not been administeredcheckpoint inhibitors as immunotherapy. Thus, the prediction isassociated with the amount of overexpression of PD-L1 mRNA in a saidsample that is identified before receiving immunotherapy with checkpointinhibitors, such as anti-PD-L1 inhibitors and may indicate if thesubject is likely to respond favourably to a treatment regimen, and canhence be used clinically to make treatment decisions by choosing themost appropriate treatment modality for any particular subject.

When assessing outcome of treatment of malignant neoplastic disease in asubject during and after immunotherapy with checkpoint inhibitors, themethod comprises the steps of:

-   -   a) performing the in vitro method for the quantification of the        PD-L1 mRNA in a sample as described herein,    -   b) determining the amount of PD-L1 mRNA in said sample,    -   c) normalizing the expression of PD-L1 with respect to B2M        expression, used as a reference gene,    -   d) comparing the amount of PD-L1 mRNA detected in said sample to        a positive control in order to estimate the overexpression of        PD-L1,    -   e) repeating steps a) to d) at one or more time points during        and after immunotherapy with checkpoint inhibitors of said        subject, and wherein a change in relative amount of PD-L1 mRNA        in a said samples over time indicates the efficacy of treatment.

The sample is advantageously obtained from a subject who is sufferingfrom a malignant neoplastic disease before and after said subject isbeing administered checkpoint inhibitors as immunotherapy. Thus, anindication of effective treatment is a relative change in decreasingamount of PD-L1 mRNA in a said sample relative a previous sampleanalyzed in the steps of repeating the method.

When assessing recurrence of malignant neoplastic disease in a subjectduring and after immunotherapy with checkpoint inhibitors, the methodcomprises the steps of:

-   -   a) performing the in vitro method for the quantification of the        PD-L1 mRNA in a sample as described herein,    -   b) determining the amount of PD-L1 mRNA in a said sample,    -   c) normalizing the expression of PD-L1 with respect to B2M        expression, used as a reference gene,    -   d) comparing the amount of PD-L1 mRNA detected in a said sample        to a positive and negative control in order to estimate the        overexpression of PD-L1,    -   e) repeating steps a) to d) at one or more time points during        and after immunotherapy with checkpoint inhibitors of said        subject, and wherein a change in relative amount of PD-L1 mRNA        in a said samples over time indicates the risk of recurrence.

Thus, an indication of recurrence is a relative change in increasingamount of PD-L1 mRNA in said samples that identify malignant neoplasticdisease during immunotherapy with anti-PD-L1 inhibitors, i.e. anover-time increase in the amount of PD-L1 mRNA in a sample relative aprevious sample analyzed in the steps of repeating the method.

The invention also relates to a kit for determining the amount of PD-L1mRNA in a sample, the kit for the quantification of PD-L1 in a samplemay also be used for:

-   -   i. determining the expression of PD-L1 mRNA in a sample, and/or    -   ii. diagnosing and/or prognosing malignant neoplastic disease in        a subject before immunotherapy with checkpoint inhibitors,        and/or    -   iii. predicting efficacy of treatment of malignant neoplastic        disease in a subject before immunotherapy with checkpoint        inhibitors, and/or    -   iv. assessing outcome of treatment of malignant neoplastic        disease in a subject during and after immunotherapy with        checkpoint inhibitors, and/or    -   v. assessing the recurrence of malignant neoplastic disease in a        subject during and after immunotherapy with checkpoint        inhibitors.

The kit may further comprise:

-   -   a forward target primer for determining the quantity of PD-L1 in        a sample, said forward target primer comprising or consisting of        at least the sequence 5′-TCATCCCAGAA-3′ (SEQ ID NO:3), more        preferably the forward target primer includes the sequence        5′-GCTGAATTGGTCATCCCAGAA-3′(SEQ ID NO: 4), and/or    -   a reverse target primer for determining the quantification of        PD-L1 in a sample, said reverse target primer comprising or        consisting of at least the sequence 5′-CATTCTCCCTT-3′ (SEQ ID        NO: 5), more preferably the reverse target primer includes the        sequence 5′-TTTCACATCCATCATTCTCCCTT-3′ (SEQ ID NO: 6), and/or    -   a hydrolysis target probe for determining the quantity of PD-L1        in a sample, said hydrolysis target probe comprising or        consisting of at least the sequence 5′-6FAM-GCACATCCTCCA-BBQ-3′        (SEQ ID NO: 7), more preferably the target hydrolysis probe        includes the sequence 5′-6FAM-ACCTCTGGCACATCCTCCAAATGAAAG-BBQ-3′        (SEQ ID NO: 8) and two fluorescent particles (FAM, BBQ), and/or    -   a forward reference primer for normalization the data for the        quantification of PD-L1 in respect to the expression of B2M as a        reference gene, said forward reference primer comprising or        consisting of at least the sequence 5′-GCCGTGTGAAC-3′ (SEQ ID        NO: 9), more preferably the forward reference primer includes        the sequence 5′-GCCTGCCGTGTGAACCATGT-3′ (SEQ ID NO: 10), and/or    -   a reverse reference primer for normalization the data for the        quantification of PD-L1 in respect to the expression of B2M as a        reference gene, said reverse reference primer comprising or        consisting of at least the sequence 5′-CTTCAAACCTC-3′ (SEQ ID        NO: 11), more preferably the reverse reference primer includes        the sequence 5′-AAATGCGGCATCTTCAAACCTC-3′ (SEQ ID NO: 12),        and/or    -   a reference hydrolysis probe for normalization the data for the        quantification of PD-L1 in respect to the expression of B2M as a        reference gene, said reference hydrolysis probe comprising or        consisting of at least the sequence 5′-6FAM-CTCGATCCCAC-BBQ-3′        (SEQ ID NO: 13), more preferably the target hydrolysis probe        includes the sequence        5′-6FAM-CATGATGCTGCTTACATGTCTCGATCCCAC-BBQ-3′ (SEQ ID NO: 14)        and two fluorescent particles (FAM, BBQ).

BRIEF DESCRIPTION OF FIGURES

FIG. 1: illustrates the procedure for the isolation of CTCs fromperipheral blood and down-stream RT-qPCR for PD-L1 and B2M.

FIG. 2: depicts the hybridization sites for primers and hydrolysis probefor the amplification of PD-L1 mRNA sequence. The primers—forward,reverse—and hydrolysis probe were depicted in bold and the exon-exonjunction was depicted with a forward slash.

FIG. 3: depicts the hybridization sites for primers and hydrolysis probefor the amplification of B2M mRNA sequence. The primers—forward,reverse—and hydrolysis probe were depicted in bold and the exon-exonjunction was depicted with a forward slash.

FIG. 4: RT-qPCR assay for PD-L1: experimental flowchart for PD-L1 mRNAexpression in clinical samples.

FIG. 5: presents the RT-qPCR calibration curves for PD-L1 and B2M(copies/4, all measured in triplicate).

FIG. 6: shows the quantitative expression of PD-L1 in fresh frozen NSCLCprimary tumors (2^(−ΔΔCq) values).

FIG. 7: shows the ΔCq values between primary tissues of NSCLC and theiradjacent normals.

FIG. 8: shows the quantitative expression of PD-L1 in CTCs (2^(−ΔΔCq)values).

FIG. 9: shows the ΔCq values between control group and CTCs isolatedfrom breast cancer metastatic samples.

DEFINITIONS

The terms used in this invention are, in general, expected to adhere tostandard definitions generally accepted by those having ordinary skillin the art of molecular biology. A few exceptions, as listed below, havebeen further defined within the scope of the present invention.

“At least one” as used herein means one or more, i.e. 1, 2, 3, 4, 5, 6,7, 8, 9, 10 etc.

As used herein “target sequence” means a sequence that is detected,amplified, both amplified and detected or is complementary to thesequences provided herein or otherwise has at least one intron in itsnative state i.e. as genomic DNA or extra chromosomal DNA. While theterm target sequence is sometimes referred to as single stranded, thoseskilled in the art will recognize that the target sequence may be doublestranded. In particular, according to this text the “target sequence” isreferred to the part of the PD-L1 sequence, which is amplified, when aprimer pair binds to the desired sequence.

As used herein, the term “primer” refers to an oligonucleotide which,produced synthetically, is capable of acting as a point of initiation ofnucleic acid synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is induced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, reverse transcriptase or thelike, and at a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency, but may alternatively be doublestranded. If double stranded, the primer is first treated to separateits strands before being used to prepare extension products. The primermust be sufficiently long to prime the synthesis of extension productsin the presence of the agents for polymerization. The exact lengths ofthe primers will depend on many factors, including temperature and thesource of primer. For example, depending on the complexity of the targetsequence, a primer typically contains 15 to 25 or more nucleotides,although it can contain fewer nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with a template. In particular, the primers, which are usedhere, are preferably designed in a way that avoids amplification ofgenomic DNA or cDNA in order to avoid non-specific amplification ofcontaminating genomic DNA in the sample.

The optional reverse transcription step in the method of the inventionis included wherever necessary in order to amplify the target sequence,i.e. when the nature of the target sequence is RNA. This process,designated reverse transcription, occurs under the direction of anRNA-dependent DNA polymerase enzyme called a reverse transcriptase. Theprocess furthermore requires buffers and reagents, such as dNTPs, forthe reverse transcription. Reverse transcription kits are commerciallyavailable and it is within the skill to perform this process. Thenucleic acid amplification reagents used in the invention includesreagents which are well known and may include, but are not limited to,an enzyme with polymerase activity e.g. heat stable polymerases such asthe Taq-polymerase (and, as necessary, reverse transcriptase activitye.g. when monitoring mRNA), enzyme cofactors such as magnesium ormanganese; salts and deoxynucleotide triphosphates (dNTPs).

The term “amplification conditions” is generally defined as conditions,which promote hybridizing or annealing of primer sequences to a targetsequence and subsequent extension of the primer sequence. It is wellknown in the art that such annealing is dependent on several parameters,including temperature, ionic strength, sequence length, complementarityand G:C content of the sequences. For example, lowering the temperaturein the environment of complementary nucleic acid sequences promotesannealing. For any given set of sequences, melt temperature, or Tm, canbe estimated by any of several known methods. Typically, diagnosticapplications utilize hybridization temperatures, which are close to(i.e. within 10° C.) the melt temperature. Ionic strength or “salt”concentration also impacts the melt temperature, since small cationstend to stabilize the formation of duplexes by negating the negativecharge on the phosphodiester backbone. Typical salt concentrationsdepend on the nature and valency of the cation but are readilyunderstood by those skilled in the art. Similarly, high G:C content andincreased sequence length are also known to stabilize duplex formationbecause G:C pairings involve 3 hydrogen bonds where A:T pairs have justtwo, and because longer sequences have more hydrogen bonds holding thesequences together. Thus, a high G:C content and longer sequence lengthsimpact the hybridization conditions by elevating the melt temperature.Once sequences are selected for a given diagnostic application, the G:Ccontent and length will be known and can be accounted for in determiningprecisely what hybridization conditions will encompass. Since ionicstrength is typically optimized for enzymatic activity the onlyparameter left to vary is the temperature. Generally, the hybridizationtemperature is selected close to or at the T_(m) of the primers orprobe. Thus, obtaining suitable hybridization conditions for aparticular primer, probe, or primer and probe set is well withinordinary skill of one practicing this art. The amplification productproduced as above can be detected during or subsequently to theamplification of the target sequence using any suitable method and aprobe disclosed in greater detail below.

As used herein, the term “melting temperature” (T_(m)) in relation to anoligonucleotide is defined as the temperature at which 50% of the DNAforms a stable double-helix and the other 50% has been separated intosingle stranded molecules. As known to those of skill in the art, PCRannealing temperature is typically a few degrees less than the T_(m),the latter of which is calculated based on oligo and salt concentrationsin the reaction.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably. “Oligonucleotide” is a term sometimes used to describea shorter polynucleotide.

The terms “hybridized” and “hybridization” refer to the base-pairinginteractions between two nucleic acids that result in formation of aduplex. It is not a requirement that two nucleic acids have 100%complementarity over their full length to achieve hybridization.

The term “external standard” as used herein means a synthetic DNA or RNAtranscript in known amount(s) or concentration(s) that is testedseparately from the test sample, i.e. through interpolation orextrapolation to a standard curve.

As used herein, “cycle threshold” (Ct) refers to quantification cyclevalues calculated from the record fluorescence measurements of the realtime quantitative PCR. “Cq” refers to the number of cycles required forthe PCR signal to reach the significant threshold. The calculated Cqvalue is proportional to the log of the number of target copies presentin the sample. The Cq quantification is performed with any method forthe real time quantitative PCR amplification described in the art[Bustin S A. Absolute quantification of mRNA using real-time reversetranscription polymerase chain reaction assays. J Mol Endocrinol 2000;25: 169-193].

“Precision” is the measure of the degree of repeatability of ananalytical method under normal operation and is normally expressed asthe percent relative standard deviation for a statistically significantnumber of samples. The two most common precision measures are“repeatability” and “reproducibility”. These are expression of twoextreme measure of precision which can be obtained. Repeatability (thesmallest expected precision) will give an idea of the sort ofvariability to be expected when a method is performed by a singleanalyst on one piece of equipment over a short time scale. If a sampleis analyzed by a number of laboratories for comparative purposes then amore meaningful precision measure to use is reproducibility (this is thelargest measure of precision).

“Limit of Detection” (LoD) is the lowest analyte concentration likely tobe reliably distinguished from the blank and at which detection isfeasible. LoD is determined by utilizing both the measured LoB and testreplicates of a sample known to contain a low concentration of analyte.

“Limit of Quantification” (LoQ) refers to the lowest concentration atwhich the analyte can not only be reliably detected but at which somepredefined goals for bias and imprecision are met. The LoQ is defined asthree times the LOD.

“Linearity” is the ability of the method to elicit test results that aredirectly proportional to analyte concentration with in a given range.Linearity is generally reported as the variance of the slope of theregression line. Traditionally linearity was regarded as a desirableproperty of methods as only linear curves could be easily interpreted.With the ready availability of computing power this is now of littleimportance and non-linear calibrations can readily be dealt with.

“False negative” refers to a test result indicates a subject does notsuffer from a malignant neoplastic disease when the subject actuallydoes have it. In the context of this application the “false negative”refers to a test result that is incorrect because the method failed todetermine the overexpression of PD-L1 in a sample from a subject.

“False positives” refers to a test result that that indicates a subjectwho suffers from a malignant neoplastic disease when the subjectactually does not have it. In the context of this application the “falsepositive” refers to a test result that is incorrect because the methodindicates to determine the overexpression of PD-L1 in a sample, whichdoes not exist.

“Healthy” refers to a subject possessing good health. Such a subjectdemonstrates an absence of any malignant or non-malignant disease. Inthe context of this application, a “healthy individual” is only healthyin that they have an absence of any malignant or non-malignant disease;a “healthy individual” may have other diseases or conditions that wouldnormally not be considered “healthy”.

“Prognosis” as used herein to refer to the prediction of the likelihoodof progression, including recurrence, metastatic spread, and drugresistance, of a malignant neoplastic disease. For example, a patienthaving an expression profile, which correlates with an invasivephenotype, may exhibit a high proliferative activity, and therefore maybe demonstrative of a favourable response to therapy, as the invasivephenotype can be a histologic characteristic used to indicate atherapy-sensitive neoplastic disease.

A “malignant” neoplasm is generally poorly differentiated (anaplasia),has characteristically rapid growth accompanied by progressiveinfiltration, invasion, and destruction of the surrounding tissue.Furthermore, a malignant neoplasm has the capacity to metastasize todistant sites. The term “metastasis” refers to the spread or migrationof cancerous cells from a primary (original) tumor to another organ ortissue, and is typically identifiable by the presence of a “secondarytumor” or “secondary cell mass” of the tissue type of the primary(original) tumor and not of that of the organ or tissue in which thesecondary (metastatic) tumor is located. For example a carcinoma of thelung that has migrated to bone is said to be metastasized lung cancer,and consists of cancer cells originating from epithelial lung cellsgrowing in bone tissue.

As used herein, the expression “clinical outcome” or “outcome” is meantto be expressed in terms of different endpoints such as Disease-FreeSurvival (DFS), Relapse-Free Survival (RFS), Time-to-Recurrence (TR),Cancer-Specific Survival (CSS) or Overall Survival (OS), in accordancewith the recommendations of Punt C J, Buyse M, Köhne C-H, Hohenberger P,Labianca R, Schmoll H J, et al. Endpoints in Adjuvant Treatment Trials:A Systematic Review of the Literature in Colon Cancer and ProposedDefinitions for Future Trials. J. Natl. Cancer Inst. 2007; 13: 998-1003.

As used herein, the expression “Relapse-Free Survival” or“Recurrence-Free Survival” (RFS) is defined as the time to any event,irrespective of the cause of this event, except for any second primarycancer. Recurrence of or death from the same cancer and alltreatment-related deaths or deaths from other causes are events. Secondprimary from the same cancers and other primary cancers are ignored, andloss to follow-up is censored.

As used herein, the expression “Disease-Free Survival” (DFS) is definedas the time to any event, irrespective of the cause of this event. Allevents are included, except loss to follow-up which is censored. As usedherein, the “Overall Survival” (OS) is defined as the time to death,irrespective of cause, whether or not the death was due to cancer. Locoregional recurrence, distant metastases, second primary cancers, andsecond other primary cancers are ignored. Loss to follow-up is censored.

“Subject” as used herein includes humans, nonhuman primates such aschimpanzees and other apes and monkey species, farm animals such ascattle, sheep, pigs, goats and horses, domestic mammals such as dogs andcats, laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be covered. In preferred embodiments, thesubject is a mammal, including humans and non-human mammals. In the mostpreferred embodiment, the subject is a human.

As used herein a “biological sample” encompasses a variety of sampletypes obtained from any subject having or not having malignant neoplasm.A typical subject is a human. For example, biological samples includesamples obtained from a tissue or blood fluids collected from anindividual suspected of having a malignant neoplasm.

“Immunotherapy” is treatment that uses certain parts of a person'simmune system to fight diseases such as cancer. This can be done eitherstimulating the immune system to work harder or smarter to attack cancercells or giving the immune system components, such as man-made immunesystem proteins. Some types of immunotherapy are also sometimes calledbiologic therapy or biotherapy. In the last few decades immunotherapyhas become an important part of treating some types of cancer.Immunotherapy includes treatments that work in different ways. Someboost the body's immune system in a very general way. Others help trainthe immune system to attack cancer cells specifically.

“Checkpoint inhibitors” (also known as immune checkpoint modulators) aredesigned to lessen the effectiveness of checkpoint proteins. They couldhave a variety of mechanisms of action, but if effective they let theimmune system see the other molecules on the surface of the cancercells. There are many kinds of immune checkpoint inhibitors, such asanti-PD-L1 inhibitors, anti-CTLA4 inhibitors etc.

As used herein the term circulating tumor cells (CTC) are cells thathave shed into the vasculature from a primary tumor and circulate in thebloodstream. CTCs thus constitute seeds for subsequent growth ofadditional tumors (metastasis) in vital distant organs, triggering amechanism that is responsible for the vast majority of cancer-relateddeaths.

As used herein, the term PD-L1 refers to the official name of the gene“Programmed death-ligand 1” (PD-L1; also called B7-H1 or CD274). The“Programmed death-ligand 1” gene is expressed on many cancer and immunecells and plays an important part in blocking the “cancer immunitycycle” by binding programmed death-1 (PD-1) and B7.1 (CD80), both ofwhich are negative regulators of T-lymphocyte activation. Binding ofPD-L1 to its receptors suppresses T-cell migration, proliferation andsecretion of cytotoxic mediators, and restricts tumour cell killing. ThePD-L1-PD-1 axis protects the host from overactive T-effector cells notonly in cancer but also during microbial infections. Blocking PD-L1should therefore enhance anticancer immunity, but little is known aboutpredictive factors of efficacy. More especially, the term “targetsequence”, as used herein refers to PD-L1 mRNA sequence.

As used herein, the term B2M refers to the official name of the gene“beta-2-microglobulin”, which encodes a serum protein found inassociation with the major histocompatibility complex (MHC) class Iheavy chain on the surface of nearly all nucleated cells. The proteinhas a predominantly beta-pleated sheet structure that can form amyloidfibrils in some pathological conditions. The encoded antimicrobialprotein displays antibacterial activity in amniotic fluid. Moreespecially, the term “reference sequence”, as used herein refers to B2MmRNA sequence.

As used herein, the term “reference gene” refers to any particular knowngenome sequence, whether partial or complete, of any organism or viruswhich may be used to reference identified sequences from a subject. Forexample, a reference genome used for human subjects as well as manyother organisms is found at the National Center for BiotechnologyInformation at www.ncbi.nlm.nih.gov. A “genome” refers to the completegenetic information of an organism or virus, expressed in nucleic acidsequences. More especially, the term “reference gene”, as used hereinrefers to B2M gene.

DETAILED DESCRIPTION

The present invention provides a highly sensitive, specific andreproducible real time RT-qPCR method for the quantification of PD-L1expression in a biological sample, such as an isolated RNA sample fromCTC in peripheral blood of patients with solid cancers or fresh frozenprimary tumor tissues. The clinical importance of PD-L1 mRNA expressionis associated with the response to targeted immunotherapies in manytypes of cancers.

Materials and Methods Patients

As a first group, a total of 31 primary NSCLC carcinomas and theircorresponding 31 adjacent non-neoplastic tissues were analyzed. As asecond group, a total of 32 peripheral blood samples were obtained from22 patients with metastatic breast cancer and 10 from healthy femaleblood donors, used as control group in order to define the specificityof the assay. The tissue samples were collected at the time of surgeryand were immediately flash frozen in liquid nitrogen and stored at −80°C. All samples were analyzed histologically to access the account oftumor component (at least 70% of tumor cells) and the quality ofmaterial (i.e., absence of necrosis).

CTC Isolation from Peripheral Blood with Positive ImmunomagneticSelection

CTC were isolated from 20 mL peripheral blood as previously described[Strati A, et al. Gene expression profile of circulating tumor cells inbreast cancer by RT-qPCR. BMC Cancer 2011; 11: 422]. This procedure isoutlined in FIG. 1. Moreover, the developed RT-qPCR assay for PD-L1 canbe applied in CTC samples isolated with different methodologies as well.To reduce blood contamination by epithelial cells from the skin, thefirst 5 ml of blood were discarded, and the collection tube was at theend disconnected before withdrawing the needle. After collection,peripheral blood was diluted with 20 mL phosphate buffered saline (PBS,pH 7.3) and peripheral blood mononuclear cells (PBMCs) were isolated bygradient density centrifugation using Ficol-Paque™ PLUS (GE Healthcare,Bio-Sciences AB) at 670 g for 30 min at room temperature. The interfacecells were removed, washed twice with 40 mL of sterile PBS (pH 7.3, 4°C.), at 530 g for 10 min, and resuspended in 1 mL of PBS. CTC wereenriched using immunomagnetic Ber-EP4 coated capture beads (Dynabeads®Epithelial Enrich, Invitrogen), according to manufacturer'sinstructions. Two fractions were isolated for each sample: theEpCAM-positive CTC fraction (CTC fraction) and the correspondingEpCAM-negative fraction containing the PBMC (PBMC fraction).

RNA Isolation from Tumor Tissues and cDNA Synthesis

Total cellular RNA was isolated with the Qiagen RNeasy Mini Reagent Set(Qiagen), according to the manufacturers' instructions. All preparationand handling steps of RNA took place in a laminar flow hood underRNase-free conditions. The isolated RNA was dissolved in RNA storagebuffer (Ambion) and stored at −70° C. until use. RNA concentration wasdetermined in the NanoDrop® ND-1000 spectrophotometer (NanoDropTechnologies). The amount of 1 μg of total RNA was used to performreverse transcription of RNA with the High-Capacity RNA-to-cDNA™ Kit(Applied Biosystems, USA) in a total volume of 20 μL, according to themanufacturer's instructions.

RNA Isolation from CTCs and cDNA Synthesis

Total RNA isolation was performed using the TRIZOL-LS reagent(Invitrogen, USA). All RNA preparation and handling steps took place ina laminar flow hood, under RNAse-free conditions. The isolated RNA wasdissolved in 20 μL of RNA storage buffer (Ambion, USA) and stored at−70° C. until use. RNA concentration was determined by absorbancereadings at 260 nm using the Nanodrop-1000 spectrophotometer (NanoDrop,Technologies, USA). mRNA was isolated from the total RNA, using theDynabeads mRNA Purification kit (Invitrogen, USA) according to themanufacturer's instructions. cDNA synthesis was performed using theHigh-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, USA) in a totalvolume of 20 μL according to the manufacturer's instructions.

Primer and Probe Designs

Primers and hydrolysis probes for PD-L1 and B2M used as reference genewere de novo in-silico designed (Table 1). In-silico design wasperformed by using Primer Premier 5.0 software (Premier Biosoft, CA,USA) to avoid primer-dimer formation, false priming sites and formationof hairpin structures. Hybridization to genomic DNA was completelyavoided. Moreover, the primers and probes were designed, so as toamplify specifically PD-L1 or B2M target genes according to the searchin the BLAST Sequence Similarity Search tool (NCBI, NIH). The hydrolysisprobes included a 5′-fluorescein (FAM) as a fluorophore covalentlyattached to the 5′-end of the oligonucleotide probe and a BlackBerry®Quencher (BBQ) as a quencher at the 3′-end. The position of primers andhydrolysis probes for each target gene are shown in FIGS. 2 and 3.

TABLE 1 RT-qPCR assay for PD-L1 and B2M expression; sequences of primersand hydrolysis probes Tm Amplicon Gene (Accession No) Sequence (5′-3′)(° C.) Size (bp) PD-L1 (NM_014143) Target forward5′-GCTGAATTGGTCATCCCAGAA-3′ 59.8 147 primer (SEQ ID NO: 4), S1Target reverse 5′-TTTCACATCCATCATTCTCCCTT-3′ 60.2 primer(SEQ ID NO: 6), S1 Hydrolysis target 5′-6FAM- 69.8 probeACCTCTGGCACATCCTCCAAATGAAAG-BBQ-3′ (SEQ ID NO: 8), S1 B2M (NM_004048)Reference forward 5′-GCCTGCCGTGTGAACCATGT-3′ 63.7  99 primer(SEQ ID NO: 10), S2 Reference reverse 5′-AAATGCGGCATCTTCAAACCTC-3′ 63.2primer (SEQ ID NO: 12), S2 Hydrolysis reference 5′-6FAM- 73.4 probeCATGATGCTGCTTACATGTCTCGATCCCAC- BBQ-3′ (SEQ ID NO: 14), S2

For each gene, a primer pair and a hydrolysis probe were designed. ForPD-L1, a primer set S1 was designed to amplify the region (147 bp) thatcomprises at least one intron-spanning site. This provides a primer pairthat will only bind to a sequence in which the introns have been splicedout, e.g. mRNA, cDNA. In particular, the forward primer is designedbetween exons 4 and 5 (intron exon junction) and the reverse primer isdesigned between exons 5 and 6 (intron exon junction) of PD-L1 mRNAsequence in order to enhance only the mRNA target sequence avoiding DNAgenomic amplification and non-specific products. The hydrolysis probe isdesigned to hybridize to an internal region within the amplicon. ForB2M, a primer pair and a hydrolysis probe were designed. For B2M, aprimer set S2 was designed to amplify the region (99 bp) that comprisesat least one intron-spanning site. This provides a primer pair that willonly bind to a sequence in which the introns have been spliced out, e.g.mRNA, cDNA. In particular, the forward primer is designed in internalregion of exon 9 of B2M mRNA sequence and the reverse primer is designedbetween exons 2 and 3 of B2M mRNA sequence for amplification only thetarget sequence avoiding non-specific products. In this case, thehydrolysis probe is designed to be hybridized between exons 3 and 4(intron exon junction). All primers and probes sequences are shown indetail in Table 1.

RT-qPCR Assay

RT-qPCR reaction was performed in the LightCycler 2.0 (IVD instrument,Roche, Germany) using glass capillary tubes (Roche Applied Science,Germany). As positive control used PBMC from healthy control samplessince PBMC express as well PD-L1. The cycling protocol was identical forboth PD-L1 and B2M and consisted of an initial 2-min denaturation stepat 95° C., followed by 45 cycles of denaturation at 95° C. for 10 s,annealing at 58° C. for 20 s, and extension at 72° C. for 20 s.Real-time-PCR was performed in a total volume of 10 μL per reaction. ThePCR reaction mix for PD-L1 and B2M are described in detail IN Table 2.

TABLE 2 Reaction components for PD-L1 and B2M RT-qPCR PD-L1 B2M ReagentsInitial conc V (μL) Final conc Initial conc V (μL) Final conc Buffer 5X3 1.5X 5X 1 0.5X Mg 25 mM 1  2.5 mM 25 mM 1.2   3 mM dNTPs 10 mM 0.2 200 μM 10 mM 0.15  150 μM BSA 10 μg/μL 0.6  0.6 μg/μL 10 μg/μL 0.3  0.3μg/μL Forward 10 μM 0.3  0.3 μM 10 μM 0.25 0.25 μM primer Reverse 10 μM0.3  0.3 μM 10 μM 0.25 0.25 μM primer Hydrolysis  3 μM 0.83 0.25 μM  3μM 0.83 0.25 μM Probe Taq  5 U/μL 0.1 0.05 U/μL  5 U/μL 0.1 0.05 U/μLpolymerase Volume of — 10 — — 10 — reaction

Example 1 Development and Analytical Validation of the Assay

The experimental flowchart of the study is outlined in FIG. 4.

Protocol Optimization.

A RT-qPCR based methods for the quantification of PD-L1 and B2Mexpression were optimized in a number of experiments, using as positivecontrol cDNA samples from peripheral blood mononuclear cells (PBMC) ofhealthy control samples and negative control of PCR reaction mix, withrespect to: PCR annealing temperature, cycling parameters, primers andprobe concentrations, Mg⁺² and dNTPs concentrations.

Single RT-qPCR was performed for PD-L1 and B2M expression.Quantification is based on real-time monitoring during PCR of labelledspecific hydrolysis probe for PD-L1. The cycle where the fluorescencesignal rises above background noise (quantification cycle, Cq) is bestquantified through the LightCycler software as the second derivativemaximum of the curve. Real-time RT-PCR for PD-L1 mRNA was performedusing the LightCycler system (Roche Diagnostics). For the developedprotocol, the primers and hydrolysis probes were used as previouslydescribed.

Real-time RT-PCR was performed in a total volume of 10 μL in theLightCycler glass capillaries. For the PCR, 1 μL of cDNA was placed intoa 9 μL reaction volume containing a PCR reaction mix, which is describedin Table 3. Moreover, the PCR conditions protocol was used as previouslydescribed.

The method can be also applied to any other real time PCR instrument,such as LightCycler 1.5 instrument, LightCycler 2.0 instrument,LightCycler 480 (Roche Diagnostics) and Applied Biosystems Real-Time PCRInstrument.

Example 2 RT-qPCR Quantification Using External Calibrators

Firstly, individual PCR amplicons specifically for PD-L1 and B2M weregenerated in order to be used as external quantification calibrators.For this purpose, total RNA was extracted from PBMC pool from normalsamples since PBMC express as well PD-L1. cDNA was synthesized andserved as a template for the amplification of PD-L1 and B2M by the abovedescribed RT-qPCR. PCR products were purified using MinElute PCRPurification Kit (Qiagen, Germany) and the amplicons were quantified inthe Nanodrop-1000 spectrophotometer (NanoDrop, Technologies, USA).

DNA concentration was converted to copies/μL by use of the Avogadronumber and the molecular weight of the amplicon number of bases of thePCR product multiplied by the mean molecular weight of a pair of nucleicacids which is 660. A standard stock solution corresponding to 10¹⁰copies/μL for each gene transcript was prepared. Serial dilutions ofthis stock amplicon solution in DNase/RNase-free water ranging from 10⁵copies/μL to 10 copies/μL served as quantification calibratorsthroughout the study. For the quantification of PD-L1 and B2M genetranscripts, an external calibration curve was obtained by plotting theconcentration of each quantification calibrator expressed as copies/μLvs the corresponding quantification cycle (Cq).

Example 3 The Limit of Detection and Linearity of Assay

The limit of detection (LOD) of the developed RT-qPCR assay for bothPD-L1 and B2M as copies/μL in the reaction was evaluated. To estimateLOD, the quantification calibrators containing a known number ofcopies/μL were prepared as described below in detail: For each genetarget a calibration curve was generated using serial dilutions of thesestandards in triplicate for each concentration, ranging from 10⁵copies/μL to 10 copies/μL. The calibration curves showed linearity from10 copies/μL up to 10⁵ copies/μL, with correlation coefficients largerthan 0.99 in both cases, indicating a precise log-linear relationship(FIG. 5). None of the primers and hydrolysis probes gave any signal forany of the gene target transcripts when 50 ng/μL of 10 different genomicDNAs was analyzed.

The LOD for both of these assays was found to be 3 copies/μL and thelimit of quantification (LOQ) was found to be 9 copies/μL, (estimatedaccording to the MIQE Guidelines, S. Bustin et. al. Clin Chem. 2009).The characteristics of the calibration curves are given below in Table3.

TABLE 3 Characteristics of the calibration curves for PD-L1 and B2MRT-qPCR PCR mean slope Intercept efficiency^(a) LOD, Gene n = 3 n = 3(%) copies/μL LOQ, copies/μL B2M −3.237 + 0.013 37.67 + 0.031 104 3copies/μL 9 copies/μL PD-L1 −3.506 + 0.14  38.43 + 0.32  93 3 copies/μL9 copies/μL ^(a)PCR efficiency is expressed as E = [10^(−1/slope)] − 1

Example 4 Evaluation of Intra and Inter-Assay Precision

Repeatability or intra-assay variance (within-run precision) of thePD-L1 RT-qPCR, was evaluated by repeatedly analyzing 3 cDNA samplescorresponding to low, medium and high mRNA expression, while for B2M wasevaluated by repeatedly analyzing 4 cDNA samples corresponding to 1, 10,100 and 1000 cells equivalents per μL of cDNA in the same assay, in 3parallel determinations.

Intra-assay variance expressed as the CVs (%) of the Cq variance forPD-L1, ranged from 0.84 to 1.2, while for B2M ranged from 0.21 to 0.72(Table 4). Intra-assay variance expressed as within-run CVs of copies/μLranged for PD-L1, from 16% to 20% and for B2M from 3.7% to 14% (Table4). Reproducibility or inter-assay variance (between-run precision) ofthe RT-qPCR assays, was evaluated by analyzing the same cDNA sample,representing 100 SKBR-3 cells for B2M and PBMCs for PD-L1 and keptfrozen in aliquots at −20° C., over a period of one month on 4 separateassays performed in 4 different days. Between-run CVs were 17% for B2M,and 15% for PD-L1 (Table 4).

TABLE 4 RT-qPCR for PD-L1 and B2M: Evaluation of intra and inter-assayprecision B2M Cq (SD) CV % Copies (SD) CV % SKBR-3 Intra-assay precision(n = 3) 1 32.56 (0.11) 0.34 3.8 (±0.29) × 10 7.9 10 29.29 (0.21) 0.723.8 (±0.55) × 10² 14 100  25.79 (0.053) 0.21 4.5 (±0.15) × 10³ 3.7 100022.40 (0.10) 0.45 4.9 (±0.36) × 10⁴ 7.3 Inter-assay precision (n = 5)100 25.73 (0.26) 1.0 4.7 (±0.85) × 10³ 17 PD-L1 Intra-assay precision (n= 3) cDNA 1 35.63 (0.30) 0.84 6.5 (±1.3) 20 cDNA 2 31.48 (0.25) 0.790.97 (±0.14) 10² 16 cDNA 3 24.45 (0.29) 1.2 1.0 (±0.18) × 10⁴ 19Inter-assay precision (n = 5) cDNA 4 29.57 (0.21) 0.71 5.32 (±0.82) ×10² 15

Example 5 Normalization of Data for the Quantification of PD-L1Expression in Primary Tumors and CTCs

The normalization of data for the quantification of PD-L1 expression hasperformed in respect to the expression of B2M as a reference gene, andusing the 2^(−ΔΔCt) method (Livak and Schmittgen, Methods 2001).

In primary tumors two samples were available for each patient: a) theprimary tumor and the corresponding non-cancerous tissue. In this casefor each sample, the expression of PD-L1 is estimated as a relativeratio to B2M used as a reference gene in both the primary tumor and itsadjacent non-cancerous tissue, used as a calibrator. Then by using the2^(−ΔΔCt) approach, the normalization of analytical signal (Cq) of PD-L1in each primary tumor sample to the corresponding analytical signal (Cq)of PD-L1 in the adjacent non-cancerous sample.

In the case of CTCs for each sample, the expression of PD-L1 isestimated as a relative ratio to B2M used as a reference gene in boththe EpCAM-positive CTC fraction and in the corresponding EpCAM-positiveCTC fraction in the group of healthy blood donors used as a calibrator.Then by using the 2^(−ΔΔCt) approach, the overexpression of PD-L1 in theEpCAM-positive CTC fraction of peripheral blood samples was evaluated.

In the EpCAM-positive CTC fraction, one sample was defined as PD-L1overexpressed based on the fold change of PD-L1 expression in respect tothe group of ten healthy individuals used as a control group. Morespecifically, a cut-off value was estimated according to the expressionof PD-L1 in the EpCAM-positive fraction of ten healthy individualsanalysed in exactly the same way as the patient's peripheral bloodsamples. For these ten control samples, the difference of the Cq value(ΔCq_(control)) for PD-L1 from the respective Cq value for B2M(Cq_(PD-L1)-Cq_(B2M)) was measured. The median value of these 10 ΔCqvalues was 15.78±0.83 (Table 5).

TABLE 5 Expression of PD-L1 in PBMC (control group). (ΔCq values:Cq_(PD-L1) − Cq_(B2M)) A/A Cq PD-L1 Cq B2M ΔCq 1 38.68 22.06 16.62 238.70 22.70 16.00 3 36.58 20.63 15.95 4 35.38 19.77 15.61 5 41.24 25.7415.50 6 40.60 23.16 17.44 7 40.40 23.81 16.59 8 38.90 24.12 14.78 939.34 22.67 16.67 10 38.93 23.99 14.94 SD 0.83 Median 15.78 Cut off14.70

Example 6 Expression of PD-L1 in Primary Tumors (NSCLC)

In this group, the quantification of PD-L1 expression was performed in31 pairs of NSCLC tissues and their adjacent non-neoplastic tissuesusing RT-qPCR. PD-L1 was expressed in all tissues, and its expressionwas normalized with respect to B2M gene expression and by using therelative quantification approach described by Livak and Schmittgen aspreviously described. In this study demonstrated that PD-L1 wasoverexpressed in 14/31 (45.2%) of NSCLC tissues (FIG. 6). The ΔCq valuesbetween primary tissues of NSCLC and their adjacent normal samples areshown in FIG. 7.

Example 7 Expression of PD-L1 Expression in CTCs

In this group, the quantification of PD-L1 in the EpCAM-positive CTCfraction (isolated from peripheral blood from metastatic breast cancerpatients) was performed. For each patient sample, the correspondingCq_(PD-L1)-Cq_(B2M) value (ΔCq_(sample)) was calculated. Then eachindividual ΔCq_(sample) value was evaluated by finding its differencefrom the median ΔCq_(control) value (ΔΔCq=ΔCq_(sample)−ΔCq_(control))(Table 6). The median value of the 2^(−ΔΔCq) for the patients groupanalysed was 2.20. The samples with a 2^(−ΔΔCq) value above 2.20 or aΔCq value below 14.7 were defined as positive for PD-L1 mRNAoverexpression (Table 6). Moreover, the ΔCq values in all of the samplesfrom healthy individuals were above 14.7 (FIG. 8).

According to the results, PD-L1 was found to be overexpressed in 8/22(36.4%) patients with verified metastasis (FIG. 8). The ΔCq valuesbetween control group and CTCs isolated from breast cancer metastaticsamples are shown in FIG. 9.

TABLE 6 Expression of PD-L1 in the EpCAM-positive CTC fraction(2^(−ΔΔCq) values) ΔCq ΔΔCq A/A Cq PD-L1 Cq B2M Cq_(PD-L1) − Cq_(B2M)ΔCq_(sample) − ΔCq_(controlmedian) 2{circumflex over ( )}^(−ΔΔCq) Result1 38.47 22.32 16.15 0.37 0.77 Negative 2 36.32 19.49 16.83 1.05 0.48Negative 3 38.71 21.31 17.40 1.62 0.33 Negative 4 37.51 21.92 15.59−0.19 1.14 Negative 5 39.24 24.24 15.00 −078 1.72 Negative 6 39.52 22.7416.78 1.00 0.50 Negative 7 34.81 20.82 13.99 −1.79 3.46 Positive 8 40.3923.2 17.19 1.41 0.38 Negative 9 38.45 21.89 16.56 0.78 0.58 Negative 1034.89 22.09 12.8 −2.98 7.89 Positive 11 36.10 21.25 14.85 −0.93 1.91Negative 12 39.30 25.16 14.14 −1.64 3.12 Positive 13 38.63 24.66 13.97−1.81 3.51 Positive 14 40.98 23.51 17.47 1.69 0.31 Negative 15 35.7821.21 14.57 −1.21 2.31 Positive 16 38.86 24.58 14.28 −1.5 2.83 Positive17 39.26 24.55 14.71 −1.07 2.10 Negative 18 38.57 21.35 17.22 1.44 0.37Negative 19 39.82 25.54 14.28 −1.5 2.83 Positive 20 39.23 20.97 18.262.48 0.18 Negative 21 38.82 21.51 17.31 1.53 0.35 Negative 22 35.3720.91 14.46 −1.32 2.50 Positive Median 2.21

Sequence Listing

1-47. (canceled)
 48. A method for either (a) assessing the outcome oftreatment of malignant neoplastic disease in a subject during and afterimmunotherapy with checkpoint inhibitors where an indication ofeffective treatment is a relative change in decreasing amount of PD-L1mRNA in a sample relative to a previous sample analyzed in the steps ofrepeating the method herein, or (b) assessing recurrence of malignantneoplastic disease in a subject during and after immunotherapy withcheckpoint inhibitors where an indication of the recurrence is an overtime increase in the amount of PD-L1 mRNA in a sample relative to aprevious sample analysed in the steps of repeating the method herein,comprising the quantification of the Programmed Death Ligand 1 (PD-L1)in a liquid biopsy sample of Circulating Tumor Cells (CTC), and saidmethod comprising: i. subjecting the liquid biopsy sample to reversetranscription using RNA present in the sample as a template in order tosynthesize a corresponding cDNA sequence, ii. forming a reaction mixturecomprising the sample, nucleic acid amplification reagents, a targetprimer pair, a target hydrolysis probe, said target primer pair andtarget hydrolysis probe being capable of hybridizing to PD-L1 mRNA, andsaid target primer pair comprising a forward target primer and a reversetarget primer wherein at least one of said forward or reverse targetprimers is capable of hybridizing to an intron spanning site of thetarget sequence of PD-L1 mRNA, iii. subjecting the reaction mixture toamplification conditions optimized to generate at least one copy of anucleic acid sequence complementary to a target sequence, and saidtarget sequence being a mRNA transcript of the PD-L1 mRNA sequence (SEQID NO: 1), iv. determining the amount of PD-L1 mRNA in the liquid biopsysample, v. normalizing the expression of PD-L1 with respect to anexpression of a reference gene whose expression levels remain constantbetween different cell types, and vi. comparing the amount of PD-L1 mRNAexpressed in the said sample to a positive control which is a samplecomprising PD-L1 mRNA and a negative control which is a sample withoutcomprising PD-L1 mRNA in order to estimate an overexpression of thePD-L1 mRNA sequence.
 49. The method according to claim 48, furthercomprising designing the forward target primer of PD-L1 to hybridizebetween exon 4 and 5 of the PD-L1 mRNA sequence.
 50. The methodaccording to claim 49, further comprising using at least a sequence5′-GTCATCCCAGAA-3′ (SEQ ID NO: 3) as the forward PD-L1 target primercomprises or using a sequence 5′-GCTGAATTGGTCATCCCAGAA-3′ (SEQ ID NO: 4)as the forward PD-L1 target primer.
 51. The method according to claim50, further comprising designing the reverse target primer of PD-L1 tohybridize between exon 5 and 6 of the PD-L1 mRNA sequence.
 52. Themethod according to claim 51, further comprising using at least asequence 5′-CATTCTCCCTT-3′ (SEQ ID NO: 5) as the reverse PD-L1 targetprimer or using a sequence 5′-TTTCACATCCATCATTCTCCCTT-3′ (SEQ ID NO: 6)as the reverse PD-L1 target primer comprises.
 53. The method accordingto claim 48, further comprising using at least a sequence5′-6FAM-GCACATCCTCCA-BBQ-3′ (SEQ ID NO: 7) as the target PD-L1hydrolysis probe.
 54. The method according to claim 53, furthercomprising using a sequence 5′-6FAM-ACCTCTGGCACATCCTCCAAATGAAAG-BBQ-3′(SEQ ID NO: 8) and two fluorescent particles as the target PD-L1hydrolysis probe.
 55. The method according to claim 53, furthercomprising selecting the reference gene from the group consisting ofhypoxanthine phosphoribosyl transferase (HPRT), β2-microglobulin (B2M),glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB), 18Sribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), α-tubulin(TUBA), albumin (ALB), ribosomal protein L32 (RPL32), TATA sequencebinding protein (TBP), cyclophilin C (CYCC), Eelongation factor 1α(EF1A), RNA polymerase II (RPII).
 56. The method according to claim 55,further comprising using β2-microglobulin (B2M) mRNA sequence (SEQ IDNO: 2) as the reference gene, using at least a sequence of5′-GCCGTGTGAAC-3′ (SEQ ID NO: 9) as the forward B2M reference primer orusing a sequence 5′-GCCTGCCGTGTGAACCATGT-3′ (SEQ ID NO: 10) as theforward B2M reference primer, and using at least the sequence of5′-CTTCAAACCTC-3′ (SEQ ID NO: 11) the reverse B2M as the referenceprimer or a sequence 5′-AAATGCGGCATCTTCAAACCTC-3′ (SEQ ID NO: 12) as thereverse B2M reference primer.
 57. The method according to 56, furthercomprising using fluorescent particles as the target or referencehydrolysis probe.
 58. The method according to claim 57, furthercomprising using a fluorescent reporter covalently attached to a 5′ endof the target or a reference hydrolysis probe and a fluorescent quencherdye attached to a 3′ end as the fluorescent particles of the target orreference hydrolysis probe.
 59. The method according to claim 58,further comprising using 6-fluorescein (FAM) as the fluorescent reporterdye and BlackBerry Quencher (BBQ) as the fluorescent quencher dye. 60.The method according to claim 56, further comprising using at least asequence of 5′-6FAM-CTCGATCCCAC-BBQ-3′ (SEQ ID NO: 13) as the referenceB2M hydrolysis probe.
 61. The method according to claim 60, furthercomprising using a sequence5′-6FAM-CATGATGCTGCTTACATGTCTCGATCCCAC-BBQ-3′ (SEQ ID NO: 14) and twofluorescent particles as the reference B2M hydrolysis probe.
 62. Themethod according to claim 48, further comprising selecting the malignantneoplastic disease from the group consisting of breast, urothelial,colorectal, esophageal, gastric, hepatocellular carcinoma, lung,melanoma, nasopharyngeal, multiple myeloma, renal cell carcinoma,lymphomas, oropharyngeal squamous cell carcinoma, cervical,glioblastoma, malignant mesotheliomas, ovarian and pancreatic cancer.63. A kit for amplifying a PD-L1 target sequence and determining theexpression of PD-L1 mRNA in a CTC sample from a human, for use in themethod according to claim 48, comprising; a) nucleic acid amplificationreagents, b) a target primer pair being capable of hybridizing to PD-L1mRNA, c) a reference primer pair being capable of hybridizing to mRNA ofa reference gene, d) a target hydrolysis probe being capable ofhybridizing to PD-L1 mRNA e) a reference hydrolysis probe being capableof hybridizing to mRNA of the reference gene, and f) instructions forperforming the in vitro method.